L-ascorbic acid is an essential dietary component for man, and is naturally present in citrus fruits and plants. It is conventionally synthesized by a variety of known methods such as that described in U.S. Pat. No. 2,265,121 to T. Reichstein using D-glucose as the starting material. Various other chemical and biological methods are known for synthesis and manufacture of L-ascorbic acid, such as those described in U.S. Pat. Nos. 2,702,808, 2,847,421 and 3,721,663, which are generally variations of the Reichstein process. However, as indicated, these are relatively complex processes which utilize glucose as starting material. Novel commercial-scale processes which utilize other starting materials would be desirable.
As described in British Patent No. 763,055, chemical-biological processes in which dehydrogenase (EC 1.3.2.3) present in enzyme animal or vegetable tissues is utilized to carry out terminal oxidation of the gamma lactones to provide L-ascorbic acid. A similar process is described in U.S. Pat. No. 4,259,443 in which hydrolyzed sugars of lactose and plant dehydrogenase enzyme (EC 1.3.2.3) derived from pea seedlings are utilized to produce L-ascorbic acid. The efficiency of the process was not disclosed but application at a commercial scale would appear to be restricted.
It has been recognized that bakers and/or brewers yeast contain L-galactono-lactone oxidase(s), an enzyme(s) believed to catalyze the terminal oxidation step in L-ascorbic acid biosynthesis in which the enzyme(s) catalyzes the oxidation of L-galactono-gamma lactone to produce L-ascorbic acid and hydrogen peroxide [Enzymologia; 31 #2 (1966), Eur. J. Biochem.; 127, 391 (1982) and others, M. Nishikimi, et al., Arch. Biochem. BiPhys., 191, 479 (1978)]. Studies of the ability of yeasts grown in a nutrient medium containing D-glucose (10%) as the carbon energy source to produce ascorbic acid analogs of the enediol class have also been carried out [Heick, et al., Can. J. Microbiol., 18, 597 (1972)]. In a similar study, Candida yeast strains have also been grown on sucrose, hexose or pentose to produce an ascorbic acid analog (D-erythroascorbic acid) [S. Murakawa, et al., Agric. Biol. Chem., 40 (6), 1255 (1976), 41 (9) 1799 (1977)]. When the yeasts were grown in the added presence of L-galactono gamma lactone, L-ascorbic acid was also identified. Although D-erythroascorbic acid was formed from a variety of carbon sources, L-ascorbic acid was only formed when the L-sugar-lactone was also present in the fermentation medium.
It is also known that a vast amount of lactose is available as a byproduct from cheese manufacture, in the form of whey, whey permeate or milk permeate. Utilization of these byproducts has long been a source of concern to cheese manufacturers.
It has also long been known that lactose obtained from whey or other fluid milk derived byproducts may be hydrolyzed to provide glucose and galactose (e.g., U.S. Pat. Nos. 2,826,502, 2,826,503, 2,749,242, 2,681,058) and it is known that whey may be fermented to provide ethanol (e.g., Food Engineering, November, 1977 pp. 74-75; British Pat. No. 1,524,618). A new process for the manufacture of L-ascorbic acid which could be adapted to utilize dairy byproduct lactose would be particularly desirable.
Accordingly, a principal object of the present invention is to provide novel bioconversion processes for producing L-ascorbic acid which may be carried out on a commercial scale. Another object of the present invention is to provide processes which may be adapted to utilize a dairy byproduct lactose source, such as whey, whey permeate or milk permeate in the manufacture of L-ascorbic acid. A still further object of the present invention is to provide microorganisms which are capable of producing L-ascorbic acid by aerobic fermentation of ethanol in the presence of various D- and L-galactose derivatives such as L-galactono-gamma-lactone. A further object is provision of fermentation media which are particularly adapted for the microbiological manufacture Of L-ascorbic acid. These and other objects will become more apparent from the accompanying drawings and the following detailed description.